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Both virus-induced asthma and enterovirus (EV) infection are common in children; however, the relationship between EV infection and virus-induced asthma has not been systematically investigated in a cohort study. This nationwide population-based cohort study investigated the association between EV infection and asthma.We used data from the Taiwan National Health Insurance Research Database. The study sample consisted of insured children who were younger than 18 years and had EV infection between 1997 and 2013 and were followed until December 2013. We identified 36,935 children with EV infection and compared them based on 36,935 age-, sex-, urbanization- and income-matched controls to analyze the risk of subsequent asthma. Cox regression analyses were performed and adjusted for sex, age, urbanization, income, preterm labor and small for gestational age, perinatal complications, allergic rhinitis, allergic conjunctivitis, atopic dermatitis and bronchiolitis.The mean follow-up interval for all patients was 8.59 years (standard deviation = 4.35 years). The mean latency period between initial EV infection and onset of asthma was 2.77 years (standard deviation = 2.43 years). EV infection was significantly associated with a higher incidence of asthma (hazard ratio = 1.65; 95% confidence interval: 1.60-1.71).A significant association was observed between EV infection and asthma in children. Health providers should be aware of the higher potential for children with EV to develop asthma in the future.

Site-directed mutagenesis (SDM) has been widely used for studying the structure and function of proteins. A one-step polymerase chain reaction (PCR)-based multiple site-directed plasmid mutagenesis method with extended non-overlapping sequence at the 3′ end of the primer increases the PCR amplification efficiency and the capacity of multi-site mutagenesis. Here, we introduced silent restriction sites in the primers used in this PCR-based SDM method by utilizing SDM-Assist software to generate mutants of Helicobacter pylori neutrophil-activating protein (HP-NAP), whose gene has low GC content. The HP-NAP mutants were efficiently generated by this modified mutagenesis method and quickly identified by a simple restriction digest due to the presence of the silent restriction site. This modified PCR-based SDM method with the introduction of a silent restriction site on the primer is efficient for generation and identification of mutations in the gene of interest.

Myotonic dystrophy type 1 (DM1) is caused by an expansion of CTG repeats in the 3' untranslated region (UTR) of the dystrophia myotonia protein kinase (DMPK) gene. Cognitive impairment associated with structural change in the brain is prevalent in DM1. How this histopathological abnormality during disease progression develops remains elusive. Nuclear accumulation of mutant DMPK mRNA containing expanded CUG RNA disrupting the cytoplasmic and nuclear activities of muscleblind-like (MBNL) protein has been implicated in DM1 neural pathogenesis. The association between MBNL dysfunction and morphological changes has not been investigated. We generated a mouse model for postnatal expression of expanded CUG RNA in the brain that recapitulates the features of the DM1 brain, including the formation of nuclear RNA and MBNL foci, learning disability, brain atrophy and misregulated alternative splicing. Characterization of the pathological abnormalities by a time-course study revealed that hippocampus-related learning and synaptic potentiation were impaired before structural changes in the brain, followed by brain atrophy associated with progressive reduction of axon and dendrite integrity. Moreover, cytoplasmic MBNL1 distribution on dendrites decreased before dendrite degeneration, whereas reduced MBNL2 expression and altered MBNL-regulated alternative splicing was evident after degeneration. These results suggest that the expression of expanded CUG RNA in the DM1 brain results in neurodegenerative processes, with reduced cytoplasmic MBNL1 as an early event response to expanded CUG RNA.

Increasing applications of fundamental biological gates have been successfully built during this decade. Proceeding to the current development, this study presents a genetic arithmetic and logical unit (ALU) design based on a series of genetic logic gates. The system level's architecture consists of three parts: temp register, ALU and accumulator register. Using the concept of the ALU on the digital computer, the authors present the fundamental function of the genetic ALU via full adder register and also build the bio-storage devices to store data generated from the genetic ALU. The system in this study shows the capability of computing and storage on the biocomputer.

Post-traumatic endocrine dysfunction is a complication of traumatic brain injury (TBI). However, there is lack of long-term follow-up and large sample size studies. This study included patients suffering from TBI registered in the Health Insurance Database. Endocrine disorders were identified using the ICD codes: 244 (acquired hypothyroidism), 253 (pituitary dysfunction), 255 (disorders of the adrenal glands), 258 (polyglandular dysfunction), and 259 (other endocrine disorders) with at least three outpatient visits within 1 year or one admission diagnosis. Overall, 156,945 insured subjects were included in the final analysis. The 1- and 5-year incidence rates of post-traumatic endocrinopathies were 0.4% and 2%, respectively. The risks of developing a common endocrinopathy (p

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established. In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin. This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

Palatoplasty can significantly decrease their middle ear re-intubation rate with a relatively lower hazard ratio compared to children who underwent VTI only.In children with cleft palate, questions remain about the overall effect of ventilation tube insertion (VTI) and palatoplasty for their OME. A large-scale study might offer more evidence for the roles of palatal surgery.This was a retrospective birth cohort study based upon a national database. We analyzed children born between 1999-2004 and diagnosed as cleft palate and/or lips. These children, according to their surgeries, were separated into two groups: (1) VTI only, and (2) VTI and palatoplasty. Kaplan-Meier analysis and log-rank test were used to calculate their cumulative tube re-insertion rates. Their hazard ratios of tube re-insertion were also analyzed.In total, 1205 cleft children were collected, with 151 in the VTI only group and 1054 in the VTI + palatoplasty group. Ventilation tube re-insertion rates were significantly lower in the VTI + palatoplasty group (p = 0.002). The cumulative re-insertion rates also showed a significant difference (p = 0.001). When compared to the VTI only group, the adjusted hazard ratio was 0.528 in the VTI + palatoplasty group (p = 0.001).

The extension of axon branches is important for target innervation but how axon branching is regulated is currently not well understood. Here, we report that Bcl11A/CTIP1/Evi9, a zinc finger transcription factor, downregulates axon branching. Knockdown of Bcl11A induced axon branching and multi-axon formation, as well as dendrite outgrowth. Due to alternative splicing, a single Bcl11A gene encodes two protein products, Bcl11A-L and -S. Bcl11A-L was found to be the main Bcl11A player in regulation of neurite arborization; Bcl11A-S is an antagonist of Bcl11A-L. Time-lapse study further suggests that Bcl11A-L knockdown enhances axon dynamics and increases the duration of axon outgrowth. Finally, the expression of DCC and MAP1b, two molecules involved in direction and branching of axon outgrowth, is controlled by Bcl11A-L. DCC overexpression rescues the phenotype induced by Bcl11A-L knockdown. In conclusion, this report provides the first evidence that Bcl11A is important for neurite arborization.

We introduce an idea of synthesizing a class of genetic registers based on the existing sequential biological circuits, which are composed of fundamental biological gates. In the renowned literature, biological gates and genetic oscillator have been unveiled and experimentally realized in recent years. These biological circuits have formed a basis for realizing a primitive biocomputer. In the traditional computer architecture, there is an intermediate load-store section, i.e. a register, which serves as a part of the digital processor. With which, the processor can load data from a larger memory into it and proceed to conduct necessary arithmetic or logic operations. Then, manipulated data are stored back to the memory by instruction via the register. We propose here a class of bio-registers for the biocomputer. Four types of register structures are presented. In silicon experiments illustrate results of the proposed design.

Background Helicobacter pylori neutrophil-activating protein (HP-NAP) is involved in H. pylori-induced gastric inflammation. Due to its immunogenic and immunomodulatory properties, HP-NAP has been used for developing vaccines against H. pylori infection and new drugs for cancer therapy. Results Here, we provide a simple process for high-yield production of HP-NAP by applying one-step negative chromatography to purify recombinant HP-NAP expressed in Escherichia coli (E. coli). In our E. coli expression system, recombinant HP-NAP constitutes nearly 70% of the total protein. Overexpressed recombinant HP-NAP is almost completely soluble upon cell lysis at pH 9.5. Under the optimal condition at pH 8.0, recombinant HP-NAP with purity higher than 95% can be obtained from E. coli by collecting the unbound fraction using diethylaminoethyl (DEAE) Sephadex resin in batch mode. The overall yield of HP-NAP from a 50-ml E. coli culture is ~19 mg. The purified HP-NAP folds into a multimer with a secondary structure of α-helix and is able to trigger the production of reactive oxygen species by neutrophils. Conclusions Purification of recombinant HP-NAP overexpressed in E. coli using DEAE Sephadex negative mode batch chromatography is an efficient method for high-yield production of highly pure HP-NAP in its native state. The purified HP-NAP is useful for various clinical applications including vaccine development, diagnosis, and new drug development.

Calcium/calmodulin-dependent serine kinase (CASK), a causative gene in X-linked mental retardation, carries out multiple functions in neurons, including vesicle trafficking of ion channels, synapse formation, and gene transcription. From a yeast two-hybrid screen, Kruppel-like zinc finger protein B cell lymphoma/COUP-TF-interacting protein 1 (Bcl11A/CTIP1) was identified as a CASK binding protein. Through alternative splicing, a single Bcl11A gene encodes two major protein products in neurons, Bcl11A-S and Bcl11A-L. CASK interacted with both Bcl11A-S and Bcl11A-L in transfected COS cells and brain. Immunofluorescence staining further indicated the colocalization of CASK and Bcl11A in the nuclei of neurons. These studies supported an interaction between CASK and Bcl11A in vivo. Bcl11A-L has previously been shown to play a role in gene transcription as well as axon outgrowth and branching. Here, we further show that Bcl11A-L rearranges the distribution of nuclear actin, which may be related to the function of Bcl11A-L in gene expression. More importantly, using cultured hippocampal neurons as a model system, we show that CASK enhances the ability of Bcl11A-L to restrict axon outgrowth and branching. Interruption of the interaction between CASK and Bcl11A increased the outgrowth and branching of axons, suggesting that the interaction between CASK and Bcl11A controls axon arborization. In conclusion, our results suggest that, through the interaction with Bcl11A, CASK plays a role in axonogenesis, which may be related to brain anatomical characteristics in humans.

J. Neurochem. (2010) 114, 1381–1392. Abstract While neuronal activity regulates neurite outgrowth and branching, the details of the underlying mechanisms are still largely unclear. This study investigated the effect of the glutamate receptors on neuritogenesis using cultured hippocampal neurons. At 3–4 days in vitro, NMDA treatment promoted axon branching but not primary axon extension. In contrast, blockade of the NMDA receptor (NMDAR) by AP5 treatment enhanced primary axon extension. NMDAR activation also increased dendrite number and total dendrite length. These results suggest that NMDAR controls axon and dendrite morphogenesis. A previous study demonstrated that knockdown of the zinc finger transcription factor B cell lymphoma 11A-long (Bcl11A-L) reduces deleted in colorectal cancer (DCC) and microtubule-associated protein (MAP) 1b expression, thereby promoting axon branching. Here, glutamate stimulation down-regulated the levels of the Bcl11A-L, DCC, MAP1b, and MAP2c proteins. Over-expression of either Bcl11A-L or DCC countered the effect of NMDA or glutamate on axon branching and dendrite outgrowth, indicating that the Bcl11A-L/DCC pathway is an important downstream effector of glutamate receptors in neurite arborization. Because knockdown of Bcl11A-L did not down-regulate MAP2c, our results suggest that glutamate receptors also use a Bcl11A-L-independent pathway to control dendrite outgrowth. To summarize, this study reveals novel pathways downstream of glutamate receptors that regulate axon and dendrite arborization.

Bcl11A/Evi9/CTIP1, a Kruppel-like zinc finger gene, plays an important role in B-cell development. In addition to expression in B lymphocytes, Bcl11A/Evi9/CTIP1 is also highly expressed in the brain, although its function there is still unclear. In the present study, regional and subcellular distributions of Bcl11A/Evi9/CTIP1 in rat brain were investigated by immunostaining and biochemical fractionation. Using antibodies recognizing the first 18 amino acid residues of Bcl11A/Evi9/CTIP1, the distribution of 2 isoforms of Bcl11A/Evi9/CTIP1 gene products, Bcl11A-L/Evi9a and Bcl11A-S/Evi9c, was examined. In rat brain, both Bcl11A-L/Evi9a and Bcl11A-S/Evi9c were expressed, although the amount of Bcl11A-S/Evi9c protein was higher. Bcl11A-S/Evi9c was widely expressed in different regions of the rat brain. In contrast, Bcl11A-L/Evi9a was more restricted, being expressed in the cerebral cortex, hippocampus, and olfactory bulb. At the subcellular level, biochemical fractionation and confocal analysis of adult rat brain revealed that, in addition to being in the nuclei of neurons, fractions of Bcl11A-L/Evi9a and Bcl11A-S/Evi9c could be found in extranuclear locations. Double staining with the synaptic marker synaptophysin indicated a synaptic distribution of Bcl11A/Evi9/CTIP1. Postsynaptic density was also biochemically purified and subjected to immunoblotting using Bcl11A/Evi9/CTIP1 antibodies. The results showed that Bcl11A-L/Evi9a was enriched in the PSD I and PSD II fractions. In contrast, only a trace amount of Bcl11A-S was detected in PSD fractions. Our study also indicated that a fraction of Bcl11A/Evi9/CTIP1 was present in the cytoplasm, even at synapses. To regulate gene expression in the nuclei, nuclear translocation of Bcl11A/Evi9/CTIP1 may be one of the mechanisms controlling neuronal Bcl11A/Evi9/CTIP1 function. © 2007 Wiley-Liss, Inc.

CASK interacting nucleosome assembly protein (CINAP) modulates gene expression and its abundance in cultured neurons is regulated by synaptic activity. To further study the function of CINAP in vivo, we examined the temporal and spatial expression profiles of CINAP. CINAP was widely expressed in different regions of adult mouse brain, including the cerebral cortex, hippocampus, striatum, hypothalamus, cerebellum, and two adult brain regions known to generate progenitor neurons. During early development, CINAP was also expressed in regions where neuronal progenitor cells were actively dividing, the ventricular and subventricular zones, suggesting that in addition to regulating gene expression in mature neurons, CINAP may also play a role in dividing cells. Since the hypothalamus responds to several physiological responses, we examined whether CINAP protein levels in the paraventricular nucleus (PVN) of the hypothalamus are regulated by changes in osmolality achieved through oral administration of hypertonic saline. Compared with control mice, mice treated with hypertonic saline expressed higher CINAP protein levels in the PVN, supporting a role of CINAP in neural response in vivo. Using confocal microscopic analysis, a significant amount of CINAP was found in the cytoplasm of neurons. Biochemical fractionation further confirmed that CINAP was associated with synapses, suggesting a translocation of CINAP protein from synapse to the nucleus. Consistent with this speculation, nuclear CINAP levels in the PVN were higher in hypertonic saline-treated mice than those who drank water. CINAP may be regulated through changes in protein stability and nuclear translocation in neurons. J. Comp. Neurol. 494:606–619, 2006. © 2005 Wiley-Liss, Inc.

CASK acts as a coactivator for Tbr-1, an essential transcription factor in cerebral cortex development. Presently, the molecular mechanism of the CASK coactivation effect is unclear. Here, we report that CASK binds to another nuclear protein, CINAP, which binds histones and facilitates nucleosome assembly. CINAP, via its interaction with CASK, forms a complex with Tbr-1, regulating expression of the genes controlled by Tbr-1 and CASK, such as NR2b and reelin. A knockdown of endogenous CINAP in hippocampal neurons reduces the promoter activity of NR2b. Moreover, NMDA stimulation results in a reduction in the level of CINAP protein, via a proteasomal degradation pathway, correlating with a decrease in NR2b expression in neurons. This study suggests that reduction of the CINAP protein level by synaptic stimulation contributes to regulation of the transcriptional activity of the Tbr-1/CASK/CINAP protein complex and thus modifies expression of the NR2b gene.

This paper presents a testable architecture for FSM synthesis. The transfer, synchronizing and distinguishing sequences are obtained simultaneously by adding extra edges, if necessary, and their associated inputs and outputs to the original FSM. The algorithm that achieves this minimizes the number of extra edges that make a machine testable. The testable machine has the following properties: (1) transfer sequences of length at most [log/sub 2/n] where n is the number of the states in the machine, to carry the machine from state S/sub i/ to state S/sub j/ for all i and j, (2) a synchronizing sequence of length at most [log/sub 2/n] which sets the machine to a specific state S1, and (3) a distinguishing sequence of length at most [log/sub 2/n]. The states can be observed at the output. Several synthesis benchmark circuits were investigated for area by using the architecture.